All publications mentioned throughout this application are fully incorporated herein by reference, including all references cited therein.
Human milk fat (HMF) is composed of about 30-40 g/L lipids. Of those, approximately 98% are triglycerides, 0.3-1% phospholipids, and 0.4% cholesterol. The phospholipids are composed of four major moieties: sphingomyelin (SM), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylinositol (PI).
Although glycerophospholipids, sphingomyelin, cholesterol and their derivatives are found in relatively small amounts in mother's milk, they play an important role in the nutrition of developing infants, and have essential roles in all physiological systems and cycles of the human body.
Total fat content increases gradually from colostrum (2.0%) through transitional (2.5% to 3.0%) to mature milk (3.5% to 4.5%) [Bitman et al. (1983) Am. J. Clin. Nutr. 38:300-312].
The role of phospholipids, and especially the role of the phospholipid backbone, in human breast milk is poorly understood. Most scientific research on human breast milk phospholipids in fact uses them as an assay for the intake and incorporation of different fatty acids on the phospholipid skeleton.
Phospholipids are involved in the structure of human milk fat globules membrane (HMFGM), representing 23% of the membrane mass. Interestingly, in contrast to the significant changes in fatty acid composition from woman to woman, related mainly to race and diet, the phospholipid composition remains constant, and is not influenced by diet. Essentially, the level of phospholipids in human breast milk only changes with the age of the infant. This further suggests that phospholipids are an essential nutritional component of human breast milk.
Phospholipids show a decrease from high levels in colostrum (1.1% out of the total fat fraction) to lower levels in mature milk (0.6%). The decline in phospholipids is consistent with an increase in the fat globule size [Ruegg et al. (1981) Biochim. Biophys. Acta 666, 7-14]. The phospholipids composition of breast milk from mothers of term and preterm infants during lactation was thoroughly studied by Bitman et al. [Bitman et al. (1984) Am. J. Clin. Nutr. 40, 1103-1119].
Milk phospholipids do not exhibit any marked differences attributable to length of gestation after day 21. This remarkable constancy in class distribution of phospholipids indicates that the composition of the membrane of the milk fat globules is identical at all stages of lactation.
The amount of phospholipids (sphingomyelin and glycerophospholipids) in human milk fat is about 15-20 mg/dL. Sphingomyelin (SM) is found at about 37% of total polar lipids, phosphatidylcholine (PC) is found at 28% of total polar lipids, phosphatidyl-ethanolamine (PE) is found to be about 19%, phosphatidylserine (PS) at 9% and phosphatidylinositol (PI) at 6%. Thus, the ratio between the polar lipids of HMF is as follows: SM>PC>PE>PS>PI.
Some glycerophospholipids, and especially those extracted from soybean, are used as dietary supplements and a variety of health benefits are associated with their intake, including improvement of cognitive functions, as well as of memory and concentration, maintenance of cellular membrane composition, and contribution to general well-being. Phospholipids and lecithins are a source of choline and they enhance the bio-availability of other nutrients and therapeutic agents.
In addition, glycerophospholipids are used as food emulsifiers, anti-oxidants, stabilizers, as well as in other food application such as mold-release and anti-caking agents. They confer unique physical properties to food products as well as personal care products, and thus are also used in pharmaceutical formulations as carriers and delivery systems.
WO 03/105609 describes a phospholipid supplement which contains PS at a concentration of at least 1% out of the total phospholipid content of the composition. Moreover, said PS is derived from soybean lecithin, rapeseed lecithin or egg yolk, and is enzymatically produced using phospholipase-D.
U.S. Pat. No. 5,709,888 presents fat mixtures comprising phospholipids and LC-PUFA, such as oleic acid, linoleic acid and alpha-linolenic acid, having an adequate level of LC-PUFA of both the n6 and n3 series.
EP 484,266 describes a mixture of phospholipids obtained from domestic animal brain sources, in addition to at least one of vegetable oil, animal fat, fish oil, and/or medium chain triglycerides, in which the ratios between LC-PUFA and phospholipids is similar to those of human milk and mediterranean diet.
Most of the sphingomyelin in milk fat is a building block of the milk fat globule membrane. Sphingomyelin is also an important building block required by the infant for the development of the brain and other tissues, as well as being important in several biochemical pathways.
Myelin is the white matter coating nerve cells, enabling them to conduct impulses between the brain and other parts of the body. It consists of a layer of proteins, packed between two layers of lipids. Myelin is produced by specialized cells: oligodendrocytes in the central nervous system, and Schwann cells in the peripheral nervous system. Myelin sheaths wrap themselves around axons, the threadlike extensions of neurons that make up nerve fibers. Each oligodendrocyte can myelinate several axons. Myelin is comprised of 80 percent lipids and 20 percent proteins. A major lipid of this important tissue is sphingomyelin. This fatty substance protects the fiber-like axons and speeds electrical signals as they travel along nerve pathways to carry out vital functions such as movement.
Different diseases and syndromes are related to disorders in myelin damage. Multiple sclerosis, for example, causes myelin to disintegrate, causing an obstruction of signal flow, which progressively leads to the loss of motor coordination and other functions.
The development of myelin sheaths and neuronal network in infants is thus crucial and depends on the availability of appropriate lipid building blocks, either from self biosynthesis or from dietary sources, i.e. human breast milk.
Sphingomyelin (SM) is composed of phosphocholine as the polar head group and sphingosine as the backbone of the molecule, and it is therefore classified as a sphingolipid. These molecules are involved in the regulation of cell growth, cell differentiation, and various other functions, including cell-substratum interactions and intracellular signal transduction. Although many foods contain a small amount of SM, its nutritional and physiologic roles have not been fully examined.
The myelin of the Central Nervous System (CNS myelin) has a higher lipid content (65-80%) than that of general cell membranes. SM and sphingolipid metabolites, such as cerebrosides and sulfatides, are prominent components of the myelin sheath. This sheath acts as an insulator for nerve impulses and controls the salutatory mode of conduction via the nodes of Ranvier. Myelination of the human CNS begins from 12 to 14 wk of gestation in the spinal cord and continues into the third decade of life in the intracortical fibers of the cerebral cortex, but the most rapid and dramatic changes occur between midgestation and the end of the second postnatal year [Brody B A et al. (1987) J. Neuropathol. Exp. Neurol. 46: 283-301; Kinney H C et al. (1988) J. Neuropathol. Exp. Neurol. 47: 217-234]. Myelination accounts for a large part of the more than tripling of brain weight that occurs during this period.
A metabolic pathway for sphingolipids has been reported [Luberto C. and Hannun Y A (1999) Lipids 34 (suppl): S5-S11]. SPT (EC 2.3.1.50) is the first step and the rate-limiting enzyme in sphingolipid biosynthesis, catalyzing the synthesis of 3-ketosphinganine from L-serine and palmitoyl-CoA. This enzyme is located in the endoplasmic reticulum or Golgi apparatus. A recent study showed that SPT activity gradually increases from the third prenatal to the third postnatal week in the hypothalamus of rats. As myelination begins at the same period in these animals, it is conceivable that an increment of SPT activity may be one of the major factors involved in myelinogenesis.
CNS myelin has a high cerebroside content when compared to its level in other tissues. Cerebroside is generated from ceramide by ceramide UDP-galactosyltransferase, which is the key enzyme in the biosynthesis of cerebrosides and catalyzes the transfer of galactose from UDP-galactose to ceramide. In rats, cerebroside is hardly detectable in the brain before 10 d after birth, but the cerebroside content increases markedly from the second to the third postnatal weeks, especially between day 14 and 23 of life. Because the period of maximum cerebroside biosynthesis corresponds with the time of most active myelination, cerebroside is generally recognized as a universal marker of CNS myelination.
Ceramides can be generated from L-serine and palmitoyl-CoA through de novo synthesis by the enzyme SPT, and from SM by sphingomyelinase. Therefore, it was hypothesized by Oshida et al. (Pediatric Research 2003, 53:589-593) that during the period of low SPT activity, cerebroside in CNS myelin of developing rats may be mainly derived from dietary SM ingested in milk that is transformed to ceramide and then to cerebroside. Oshida et al. have shown that when the activity of SPT was inhibited through the administration of an appropriate inhibitor, causing a decrease of cerebrosides in rat CNS myelin, the normal maturation and weight of the myelin tissue could be maintained through the administration of dietary SM. Dietary supplementation of SM restored the brain weight and myelin dry weight that were decreased by the SPT inhibitory treatment. Furthermore, electron microscopy showed that the axon diameter of the inhibited group was restored following the introduction of dietary SM. These findings suggest that orally ingested SM is transformed to ceramide or other metabolites in the intestinal tract, which are absorbed from the bowel and entered the circulation to reach the CNS across the blood-brain barrier.
Although sphingomyelin has been thought to be an inert constituent of cell membranes, current studies suggest that metabolites of sphingomyelin are involved in signal transduction pathways. SM plays a key role in the regulation of cellular processes. Dietary SM can contribute to myelination of developing CNS and protect against toxic and inhibitory conditions. SM is also a building block to other lipids, such as ceramides.
Breast-feeding seems to contribute to rapid growth of brain weight, which is mostly the result of myelination. At birth there is very little myelin, but by 3 years, most axons have myelin coatings.
Since the human milk fat globule membrane resembles the structure and/or function of cell membranes, it is curious that its level of SM is so high, suggesting that the presence of SM can be attributed not just to its role of membrane building block, but also as a dietary source for SM. This role is of great importance at early stages of gestation in order to provide SM for myelin build-up prior to the completion of the biosynthetic route of cerebrosides, as described above. Indeed, the higher levels of phospholipids in general, and of SM in particular, at the early stages of gestation may not be coincidental but rather required to supply dietary SM to compensate the just developing biosynthetic machinery of cerebrosides.
Sphingomyelin is usually not produced on commercial levels, and it is produced only from animal sources, such as bovine milk, eggs, or animal brain. Animal sources, especially those related to brain tissues, are of course avoided in infant nutrients due to the risk of prion disease. In most cases, sphingomyelin of animal sources is produced at high levels of purity, mainly for purposes of analytical standards and for scientific research. These high purity sphingomyelin preparations are characterized by their extremely high cost and scarce availability, and thus are also not feasible for general popular consumption.
Recently, the dairy industry has started to utilize dairy waste to produce nutritional preparations which contain milk proteins, carbohydrates and small amounts of lipids. The latter include neutral lipids as well as polar lipids, including glycerophospholipids as well as sphingolipids, among them sphingomyelin. These preparations contain extremely low levels of sphingomyelin and phosphatidylserine, making them incompatible as an industrial source for these nutrients. A typical preparation (e.g. SM3 Powder, produced by S.A. Corman of Belgium), contains about 4% w/w of PC, 3.2% w/w of PE, 1.6% w/w of PS, 0.9% w/w of PI and about 2.6% w/w of SM. Such low levels would entail use of very large quantities of such preparations in order to deliver even small amount of PS and sphingomyelin. Furthermore, such large quantities would result in the delivery of non-required and non-desired proteins and carbohydrates, the latter mainly in the form of lactose.
The above described commercial milk lipids preparations, although having some similarity to HMF polar lipids, still differ from the latter. The ratio between the polar lipids in the above commercial milk-derived preparations is PC>PE>SM>PS>PI, while in HMF, the ratio between the polar lipids is SM>PC>PE>PS>PI. Particularly, in HMF the level of sphingomyelin is always higher than that of PC, the ratio being of about 1.3, while in the above commercial milk-derived preparations, this ratio is about 0.65.
Rombaut and colleagues provide phospholipid compositions of several dairy products [Rombaut et al. (2005) J. Dairy Sci. 482:488]. None of the tested dairy products gave a polar lipids ratio that is corresponding to the SM>PC>PE>PS>PI ratio of found in HMF.
As can be seen in Table 1, the lipid composition of a typical commercial milk-derived preparation differs from HMF mainly in the level of sphingomyelin, which is lower than the level of PC, and in the higher level of PE.
TABLE 1LipidCommercial milk lipidsHMF lipidsclass(% from total polar lipids)(% from total polar lipids)SM21.137.5PC32.528PE26.019.5PS13.09PI7.46Total100100
In WO 2005/051091, the present inventors described a composition that mimics the phospholipid composition of human breast milk. The present invention concerns the polar lipids of human breast milk and the importance of their supplementation by other sources in infant as well as in adult nutrition. Thus, it is an aim of the present invention to provide lipid preparations, particularly cost effective preparations, with high levels of cerebral-like lipids for advanced infant nutrition and for use in dietary supplements, functional foods and pharmaceutical compositions for promoting brain health.
WO 2005/051091 describes lipid preparations mimicking the polar lipid composition of human breast milk fat (HMF), which includes glycerophospholipids such as PC, PE, PS, and PI as well as other polar lipids, such as sphingomyelin. These lipid preparations are essentially obtained from mixtures of vegetable-derived phospholipids, preferably soybean, as well as structured phospholipids, such as trans-phosphatidylated lecithins. Other lipid preparations mimicking the polar lipids of HMF described in said publication comprise bovine milk-derived sphingomyelin. In that earlier application, the inventors used pure bovine milk sphingomyelin, obtainable as an analytical standard or research chemical, which is not particularly suitable for use in infant nutrition or dietary supplements due to its high cost and extremely low availability, as mentioned previously.
Thus, it is a purpose of the current invention to provide polar lipid preparations mimicking the polar lipids of HMF, optionally comprising SM, wherein the source of said polar lipids is a natural non-brain lipid source.
It is another object of the present invention to provide a dietary supplement which guarantees the sufficient and recommended intake of phospholipids, especially of PS and sphingomyelin, in the form of a mimetic substitute of the phospholipids from human breast milk lipid, aimed especially for infants and young children consumption, as well as pregnant women. Other uses and objects of the invention will become apparent as the description proceeds.